牛传染性鼻气管炎病毒gD基因截短表达及其抗血清制备  

Characterization of Truncated Glycoprotein D of Infectious Bovine Rhinotracheitis Virus Expressed in E.coli and Its Antiserum Preparation

作　　者：张辉[1,2] 宋玲玲[2] 杨宏军[2] 王洪梅[2] 武建明[2] 候佩莉[2] 何洪彬[2] 王立群[1] 

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150030 [2]山东省农业科学院奶牛研究中心,山东济南250100

出　　处：《家畜生态学报》2013年第1期29-33,共5页Journal of Domestic Animal Ecology

基　　金：泰山学者海外特聘专家专项;国家奶牛产业技术体系建设专项;国家自然科学基金(31272586);国家转基因重大专项(2009ZX08007-006B;2011ZX08007-002;2011ZX08008-004);济南市高校院所主创新计划(201004027;201202059;201102034)

摘　　要：gD糖蛋白是IBRV感染细胞的主要分子,在病毒吸附和侵入宿主细胞中发挥着重要作用。利用IBRV基因组DNA为模板,经生物学软件DNAstar分析主要抗原区,PCR扩增gD基因786bp片段,定向连接到pET32a(+)表达载体中,筛选阳性克隆转化BL21(DE3)表达菌中,IPTG诱导表达部分可溶的重组蛋白。Ni-NTA非变性纯化系统纯化蛋白,纯化的蛋白免疫新西兰兔制备抗血清,利用间接ELISA法检测抗血清效价为1∶6400;同时以该血清进行血清中和病毒实验,中和指数为1∶640,显示该抗血清具有良好的抗病毒能力,为进一步开发牛传染性鼻气管炎(IBR)的诊断检测试剂和疫苗奠定了重要基础。GD glycoprotein is the main molecular of which IBRV infect cells ,and it plays an important role in absorbing and invading of host cell. Using IBRV genomic DNA as the template,a 786 bp fragment of glycoprotein D gene was amplified by PCR with specific primers. The PCR product was cloned into pET32a （＋） vector and expressed in partially soluble form in E. coli after induction of cultured E. coli BL21（DE3） with IPTG. The fusion protein was purified by immobilized Ni-NTA resin under native conditions. Immu- nize the New Zealand rabbits with purified protein so as to prepare the antisera;The titer of the rabbit anti- body which prepared by pET32a-gD was 1 ：6 400;The result of serum neutralization test is 1 ： 640, this antiserum shows good anti-virus ability. This research will lay a foundation for further studies on diagnos- tic reagent and bacterin of IBR.

关 键 词：牛传染性鼻气管炎病毒 gD糖蛋白 原核表达 抗血清 

分 类 号：S811.6[农业科学—畜牧学]